The seminal discovery by Kohler and Milstein (Kohler, G. and Milstein, C., 1975) of mouse “hybridomas” capable of secreting specific monoclonal antibodies (mAbs) against predefined antigens ushered in a new era in experimental immunology. Many problems associated with antisera were circumvented. Clonal selection and immortality of hybridoma cell lines assured monoclonality and permanent availability of antibody products. At the clinical level, however, the use of such antibodies is clearly limited by the fact that they are foreign proteins and act as antigens in humans.
Since the report of Kohler and Milstein (Kohler, G. and Milstein, C., 1975), the production of mouse monoclonal antibodies has become routine. However, the application of xenogenic monoclonal antibodies for in vivo diagnostics and therapy is often associated with undesirable effects such as a human anti-mouse immunoglobulin response. In addition, monoclonal antibodies have great potential as tools for imaging. Moreover, therapeutic treatment has motivated the search for means for the production of human monoclonal antibodies (humAbs)(Levy, R., and Miller R A., 1983).
However, progress in this area has been hampered by the absence of human myelomas suitable as fusion partners with characteristics similar to those of mouse myeloma cells (Posner M R, et al., 1983). The use of Epstein-Barr virus (EBV) has proved to be quite efficient for human lymphocyte immortalization (Kozbor D, and Roder J., 1981; Casual O, 1986), but has certain limitations such as low antibody secretion rate, poor clonogenicity of antibody-secreting lines, and chromosomal instability requiring frequent subcloning. Undifferentiated human lymphoblastoid cell lines appear more attractive. In contrast to differentiated myeloma cells, these cell lines are readily adapted to culture conditions, though the problems of low yield and unstable secretion remain unresolved (Glassy M C, 1983; Ollson L, et al., 1983). The best potential fusion partners are syngenic myeloma cells with well-developed protein synthesis machinery (Nilsson K. and Ponten J., 1975). However, due to culturing difficulties few lines have been conditioned for in vitro growth and capability to produce viable hybrids (Goldman-Leikin R E, 1989). Existing myelomas have low fusion yield and slow hybrid growth, although monoclonal antibody production is relatively stable (Brodin T, 1983). Genetic instability is a major disadvantage of interspecies hybrids. This is the case, for example, when a mouse myeloma is used as the immortalizing partner. Production of mouse-human cell hybrids is not difficult, and these cells have growth characteristics In vitro similar to those of conventional mouse-mouse hybridomas (Teng N N H, 1983). However, spontaneous elimination of human chromosomes considerably reduces the probability of stable mAb secretion (Weiss M C, and Green H., 1967). In order to improve growth characteristics and stability of human monoclonal antibody production, heterohybrids between mouse myeloma cells and human lymphocyte (Oestberg L, and Pursch E., 1983) as well as heteromyelomas (Kozbor D, et. al., 1984) are used as fusion partners.
The role of humoral immunity in cancer is poorly understood. Numerous data demonstrate the presence of tumor specific, anti-tumor antibodies in cancer patients. Such antibodies can participate in potential protective anti-tumor responses that can eliminate tumor cells through any of several physiological mechanisms. Anti-tumor antibodies developed in the laboratory through immunization of animals bearing malignant tissues offer great promise in diagnostics and imaging, but have serious shortcomings in clinical application because such antibodies themselves can provoke strong immune reactions and lack important biological functions. Until recently, fully human antibodies directed to tumor-associated antigens have not been available because the human fusion partner cell lines necessary to construct human hybridomas capable of making human antibodies in large quantities were not adequate.
The general idea of developing fully human monoclonal antibodies using B-lymphocytes directly from cancer patients was discussed a few years ago. However the implementation of this idea became possible only recently when the appropriate fusion partner cell line was developed. It is now possible to capture specific B-lymphocytes producing such antibodies and maintain them in culture, harvesting the antibodies of interest.
The present invention comprises a unique fusion partner cell line that fuses with human lymphocytes derived from lymph nodes, spleen, tonsils, or peripheral blood. The cell line allows for immortalization of cancer-specific B-cells through hybridoma technique. The resulting hybrids have proved to be stable producers of human immune substances called immunoglobulins and represent a reliable source of human antibodies for immunotherapy. Using a proprietary fusion partner cell line, which was designated as MFP-2, a few human antibody-producing hybridomas with specificity towards human breast and prostate cancer were established, and thereby several monoclonal antibodies with specific immunoreactivity towards human breast and prostate cancer were developed. These antibodies reacted both with the human cancer cell lines and with primary tumor tissues. These fully human antibodies have specificity to human cancer cell lines as well as primary cancer tissues. Antigen targets were identified for some of these antibodies. Also developed was a hybridoma fusion system, which allows for capturing human lymph node or peripheral blood lymphocytes secreting specific antibodies to cancer antigens. These fully human antibodies may be used to help identify novel tumor-associated antigens, or may be employed for in vivo diagnostic and immunotherapeutic treatment of cancer.
Potential advantages of human monoclonal antibodies include the possibility of identifying the molecular target of the antibody. Such a target could turn out to be a novel molecule altogether or a known molecule whose association with cancer is novel itself. A few years ago scientists at the Ludwig Institute for Cancer Research developed the SEREX method, which allows the identification of novel tumor-associated antigens through the spontaneous antibodies present in cancer patients' blood. Their task was focused specifically on the identification of novel tumor markers. The present invention focused initially on the development of human monoclonal antibodies capable of differentiating cancerous from normal tissue. The identity of a molecular target was secondary to this mission.
In the present invention, molecular targets for some of the antibodies were identified and shown to be specific only for cancer cells. One of the targets which appeared is the PDZ domain containing protein localized both in cytosol and cell membrane of human breast cancer cells. This protein, called GIPC or TIP-2 (Tax interacting protein clone 2) is involved in vesicle trafficking and formation of protein networks. It has several properties, such as the ability to bind to RGS-Ga interacting protein, C domain, binding to HTLV-1 oncogene tax and binding both to a-actinin and glucose transporter 1. The precise physiological role of this protein is not known, while it shows a consistent overexpression in breast cancer cells, with negligible if any expression in prostate cancer cells and none in human fibroblasts. Although this protein was described previously (2), its association with cancer was not known. It was also not known that a spontaneous antibody response to this marker occurs in breast cancer patients.
One advantage of the present invention is that establishing the association of TIP-2 with malignant transformation allows application of this antigen/protein as a diagnostic marker, both in vitro and in vivo, for immunohistopathology analysis as well as for immunochemical testing; This protein may be found in the circulation in cancer patients. This protein could also serve as a molecular target for therapeutic purposes given its specific expression in primary tumors. This protein can also be used as a soluble tumor marker for cancer diagnostic, cancer progression and monitoring of cancer treatment in breast and prostate cancer patients. Since this protein is expressed on the surface of cancer cells, it can be used as a target for the specific antibody-driven delivery of liposomes loaded with drugs, or antibody-conjugated drugs, prodrugs, toxins or inhibitors of cell growth. Proving the relevance of TIP-2 for cell survival, this novel marker can be considered as a candidate for vaccine development for immunotherapy of cancer.
Antibodies to TIP-2 derived from breast cancer patient's lymphocytes can be used as a vector for in vivo diagnostic (imaging) and immunotherapy (e.g., for delivery of drug-loaded liposomes, or radioimmune- or immunotoxic conjugates to the tumor site). Fully human monoclonal antibodies to TIP-2 can and will be used to isolate preparative quantities of TIP-2 from breast cancer cells or primary tumors and to develop high affinity mouse antibodies for the purpose of diagnostic and therapeutic use had their biological value been proven. The present invention also provides a basis for the possible development of specific immunoassays or an immunohistochemistry kit for the detection and measurement of this novel tumor marker.
An advantage of the present invention is that human antibodies directed to TIP-2 can be used as an immunosorbent tool for isolation and further characterization of this protein's chemical structure (amino acid composition, protein sequence, modification).
Another advantage of the present invention is an immunosorbent prepared on the basis of human anti-TIP-2 monoclonal antibodies allows isolation of this antigen and its use for developing mouse monoclonal antibodies of high affinity and specificity which can be used to develop better tools for TIP-2 immunoassay.
Another advantage of the present invention is that, knowing the DNA sequence for TIP-2 and its association with cancer, it becomes possible to screen different tissues, normal as well as cancerous, for the expression of this marker.
Another advantage of the present invention is, since human monoclonal antibodies to TIP-2 are available and there is a strong potential to develop non-human antibodies which are even more efficient for certain diagnostic and therapeutic purposes, it is highly likely that TIP-2 can be used as a potential target for immunotherapy and for in vivo diagnostic (imaging).
Another advantage is that since TIP-2 was identified through naturally developed antibodies in breast cancer patients, its existence supports the hypothesis that this antigen can be immunogenic in humans and hence can be considered as a starting candidate for the development of an anti-cancer vaccine.